Characterisation of microvasulature changes after closed soft tissue trauma by contrast enhanced micro-CT imaging

INTRODUCTION It is known that the vascular morphology and functionality are changed following closed soft tissue trauma (CSTT) [1], and bone fractures [2]. The disruption of blood vessels may lead to hypoxia and necrosis. Currently, most clinical methods for the diagnosis and monitoring of CSTT with or without bone fractures are primarily based on qualitative measures or practical experience, making the diagnosis subjective and inaccurate. There is evidence that CSTT and early vascular changes following the injury delay the soft tissue tissue and bone healing [3]. However, a precise qualitative and quantitative morphological assessment of vasculature changes after trauma is currently missing. In this research, we aim to establish a diagnostic framework to assess the 3D vascular morphological changes after standardized CSTT in a rat model qualitatively and quantitatively using contrast-enhanced micro-CT imaging. METHODS An impact device was used for the application of a controlled reproducible CSTT to the left thigh (Biceps Femoris) of anaesthetized male Wistar rats. After euthanizing the animals at 6 hours, 24 hours, 3 days, 7 days, or 14 days after trauma, CSTT was qualitatively evaluated by macroscopic visual observation of the skin and muscles. For visualization of the vasculature, the blood vessels of sacrificed rats were flushed with heparinised saline and then perfused with a radio-opaque contrast agent (Microfil, MV 122, Flowtech, USA) using an infusion pump. After allowing the contrast agent to polymerize overnight, both hind-limbs were dissected, and then the whole injured and contra-lateral control limbs were imaged using a micro-CT scanner (µCT 40, Scanco Medical, Switzerland) to evaluate the vascular morphological changes. Correlated biopsy samples were also taken from the CSTT region of both injured and control legs. The morphological parameters such as the vessel volume ratio (VV/TV), vessel diameter (V.D), spacing (V.Sp), number (V.N), connectivity (V.Conn) and the degree of anisotropy (DA) were then quantified by evaluating the scans of biopsy samples using the micro-CT imaging system. RESULTS AND DISCUSSION A qualitative evaluation of the CSTT has shown that the developed impact protocols were capable of producing a defined and reproducible injury within the region of interest (ROI), resulting in a large hematoma and moderate swelling in both lateral and medial sides of the injured legs. Also, the visualization of the vascular network using 3D images confirmed the ability to perfuse the large vessels and a majority of the microvasculature consistently (Figure 1). Quantification of the vascular morphology obtained from correlated biopsy samples has demonstrated that V.D and V.N and V.Sp were significantly higher in the injured legs 24 hours after impact in comparison with the control legs (p<0.05). The evaluation of the other time points is currently progressing. CONCLUSIONS The findings of this research will contribute to a better understanding of the changes to the vascular network architecture following traumatic injuries and during healing process. When interpreted in context of functional changes, such as tissue oxygenation, this will allow for objective diagnosis and monitoring of CSTT and serve as validation for future non-invasive clinical assessment modalities.

A novel characterisation model for microvasulature changes following closed soft tissue trauma using micro-CT imaging

INTRODUCTION There is evidence that the reduction of blood perfusion caused by closed soft tissue trauma (CSTT) delays the healing of the affected soft tissues and bone [1]. We hypothesise that the characterisation of vascular morphology changes (VMC) following injury allows us to determine the effect of the injury on tissue perfusion and thereby the severity of the injury. This research therefore aims to assess the VMC following CSTT in a rat model using contrast-enhanced micro-CT imaging. METHODOLOGY A reproducible CSTT was created on the left leg of anaesthetized rats (male, 12 weeks) with an impact device. After euthanizing the animals at 6 and 24 hours following trauma, the vasculature was perfused with a contrast agent (Microfil, Flowtech, USA). Both hind-limbs were dissected and imaged using micro-CT for qualitative comparison of the vascular morphology and quantification of the total vascular volume (VV). In addition, biopsy samples were taken from the CSTT region and scanned to compare morphological parameters of the vasculature between the injured and control limbs. RESULTS AND DISCUSSION While the visual observation of the hindlimb scans showed consistent perfusion of the microvasculature with microfil, enabling the identification of all major blood vessels, no clear differences in the vascular architecture were observed between injured and control limbs. However, overall VV within the region of interest (ROI)was  measured to be higher for the injured limbs after 24h. Also, scans of biopsy samples demonstrated that vessel diameter and density were higher in the injured legs 24h after impact. CONCLUSION We believe these results will contribute to the development of objective diagnostic methods for CSTT based on changes to the microvascular morphology as well as aiding in the validation of future non-invasive clinical assessment modalities.

Can vasculature changes be characterised morphologically after closed soft tissue trauma?

INTRODUCTION Closed soft tissue trauma (CSTT) can be the result of a blunt impact, or a prolonged crush injury and involves damage to the skin, muscles and the neurovascular system. It causes a variety of symptoms such as haematoma and in severe cases may result in hypoxia and necrosis. There is evidence that early vasculature changes following the injury delays the tissue healing [1]. However, a precise qualitative and quantitative morphological assessment of vasculature changes after trauma and the effect of this on CSTT healing is currently missing. Research aims: Developing an experimental rat model to characterise the structural changes to the vasculature after trauma qualitatively and quantitatively using micro CT. MATERIAL AND METHODS An impact device was developed to apply a controlled reproducible CSTT to the left thigh (Biceps Femoris) of anaesthetised rats [3]. After euthanizing the animals at 6 hours after trauma, CSTT was qualitatively evaluated by macroscopic observations of the skin and muscles. For vasculature visualisation, the blood vessels of sacrificed rats were flushed with heparinised saline and then perfused with a radio-opaque contrast agent (Microfil) using an infusion pump (Figure 4). The overall changes to the vasculature as a result of impact trauma were characterised qualitatively based on the 3D reconstructed images of the vasculature (Figure 5). For a smaller region of interest, the morphological parameters such as vessel thickness (diameter), spacing, and average number per volume were quantified using the scanner’s software. RESULTS AND DISCUSSION Visual observation of CSTT has revealed a haematoma in some animals (Figure 3). Micro CT images indicate good perfusion of the vasculature with contrast agent, allowing the major vessels to be identified (Figure 5). Qualitatively and quantitatively, no differences between injured and non-injured legs were observed at 6 h after trauma. Further time points of 12h, 24h, 3 days and 14 days after trauma will be characterised for identifying temporal changes of the vasculature during healing. Histomorphometical studies are required for validation of the results derived from the micro CT imaging. CONCLUSION AND FUTURE DIRECTION Findings of this research may contribute towards the establishment of a fundamental basis for the quantitative assessment and monitoring of CSTT based on microvasculature changes after trauma, which will ultimately allow for optimising the clinical treatment and improve patient outcomes.

Visual function and CFH/ARMS2 risk genotypes in macular dystrophy due to maternally inherited diabetes and deafness

Maternally inherited diabetes and deafness (MIDD) is an autosomal dominant inherited syndrome caused by the mitochondrial DNA (mtDNA) nucleotide mutation A3243G. It affects various organs including the eye with external ophthalmoparesis, ptosis, and bilateral macular pattern dystrophy.1, 2 The prevalence of retinal involvement in MIDD is high, with 50% to 85% of patients exhibiting some macular changes.1 Those changes, however, can vary between patients and within families dramatically based on the percentage of retinal mtDNA mutations, making it difficult to give predictions on an individual’s visual prognosis...

Prostacyclin synthase expression and epigenetic regulation in nonsmall cell lung cancer

BACKGROUND: Prostacyclin synthase (PGIS) metabolizes prostaglandin H(2), into prostacyclin. This study aimed to determine the expression profile of PGIS in nonsmall cell lung cancer (NSCLC) and examine potential mechanisms involved in PGIS regulation. METHODS: PGIS expression was examined in human NSCLC and matched controls by reverse transcriptase polymerase chain reaction (RT-PCR), Western analysis, and immunohistochemistry. A 204-patient NSCLC tissue microarray was stained for PGIS and cyclooxygenase 2 (COX2) expression. Staining intensity was correlated with clinical parameters. Epigenetic mechanisms underpinning PGIS promoter expression were examined using RT-PCR, methylation-specific PCR, and chromatin immunoprecipitation analysis. RESULTS: PGIS expression was reduced/absent in human NSCLC protein samples (P <.0001), but not mRNA relative to matched controls. PGIS tissue expression was higher in squamous cell carcinoma (P =.004) and in male patients (P <.05). No significant correlation of PGIS or COX2 expression with overall patient survival was observed, although COX2 was prognostic for short-term (2-year) survival (P <.001). PGIS mRNA expression was regulated by DNA CpG methylation and histone acetylation in NSCLC cell lines, with chromatin remodeling taking place directly at the PGIS gene. PGIS mRNA expression was increased by both demethylation agents and histone deacetylase inhibitors. Protein levels were unaffected by demethylation agents, whereas PGIS protein stability was negatively affected by histone deacetylase inhibitors. CONCLUSIONS: PGIS protein expression is reduced in NSCLC, and does not correlate with overall patient survival. PGIS expression is regulated through epigenetic mechanisms. Differences in expression patterns between mRNA and protein levels suggest that PGIS expression and protein stability are regulated post-translationally. PGIS protein stability may have an important therapeutic role in NSCLC. © 2011 American Cancer Society.

A biomimetic extracellular matrix for cartilage tissue engineering centered on photocurable gelatin, hyaluronic acid and chondroitin sulfate

The development of hydrogels tailored for cartilage tissue engineering has been a research and clinical goal for over a decade. Directing cells towards a chondrogenic phenotype and promoting new matrix formation are significant challenges that must be overcome for the successful application of hydrogels in cartilage tissue therapies. Gelatin-methacrylamide (Gel-MA) hydrogels have shown promise for the repair of some tissues, but they have not been extensively investigated for cartilage tissue engineering. We encapsulated human chondrocytes in gel-MA based hydrogels, and show that with the incorporation of small quantities of photo-crosslinkable hyaluronic acid methacrylate (HA-MA), and to a lesser extent chondroitin sulfate methacrylate (CS-MA), chondrogenesis and mechanical properties can be enhanced. The addition of HA-MA to Gel-MA constructs resulted in more rounded cell morphologies, enhanced chondrogenesis as assessed by gene expression and immunofluorescence, and increased quantity and distribution of the newly synthesised ECM throughout the construct. Consequently, while the compressive moduli of control Gel-MA constructs increased by 26 kPa after 8 weeks culture, constructs with HA-MA and CS-MA increased by 96 kPa. The enhanced chondrogenic differentiation, distribution of ECM, and improved mechanical properties make these materials potential candidates for cartilage tissue engineering applications.

Development and validation of anthropometric prediction equations for estimation of lean body mass and appendicular lean soft tissue in Indian men and women

Lean body mass (LBM) and muscle mass remains difficult to quantify in large epidemiological studies due to non-availability of inexpensive methods. We therefore developed anthropometric prediction equations to estimate the LBM and appendicular lean soft tissue (ALST) using dual energy X-ray absorptiometry (DXA) as a reference method. Healthy volunteers (n= 2220; 36% females; age 18-79 y) representing a wide range of body mass index (14-44 kg/m2) participated in this study. Their LBM including ALST was assessed by DXA along with anthropometric measurements. The sample was divided into prediction (60%) and validation (40%) sets. In the prediction set, a number of prediction models were constructed using DXA measured LBM and ALST estimates as dependent variables and a combination of anthropometric indices as independent variables. These equations were cross-validated in the validation set. Simple equations using age, height and weight explained > 90% variation in the LBM and ALST in both men and women. Additional variables (hip and limb circumferences and sum of SFTs) increased the explained variation by 5-8% in the fully adjusted models predicting LBM and ALST. More complex equations using all the above anthropometric variables could predict the DXA measured LBM and ALST accurately as indicated by low standard error of the estimate (LBM: 1.47 kg and 1.63 kg for men and women, respectively) as well as good agreement by Bland Altman analyses. These equations could be a valuable tool in large epidemiological studies assessing these body compartments in Indians and other population groups with similar body composition.

Design, construction, and analysis of cell line arrays and tissue microarrays for gene expression analysis

Cell line array (CMA) and tissue microarray (TMA) technologies are high-throughput methods for analysing both the abundance and distribution of gene expression in a panel of cell lines or multiple tissue specimens in an efficient and cost-effective manner. The process is based on Kononen's method of extracting a cylindrical core of paraffin-embedded donor tissue and inserting it into a recipient paraffin block. Donor tissue from surgically resected paraffin-embedded tissue blocks, frozen needle biopsies or cell line pellets can all be arrayed in the recipient block. The representative area of interest is identified and circled on a haematoxylin and eosin (H&E)-stained section of the donor block. Using a predesigned map showing a precise spacing pattern, a high density array of up to 1,000 cores of cell pellets and/or donor tissue can be embedded into the recipient block using a tissue arrayer from Beecher Instruments. Depending on the depth of the cell line/tissue removed from the donor block 100-300 consecutive sections can be cut from each CMA/TMA block. Sections can be stained for in situ detection of protein, DNA or RNA targets using immunohistochemistry (IHC), fluorescent in situ hybridisation (FISH) or mRNA in situ hybridisation (RNA-ISH), respectively. This chapter provides detailed methods for CMA/TMA design, construction and analysis with in-depth notes on all technical aspects including tips to deal with common pitfalls the user may encounter. © Springer Science+Business Media, LLC 2011.

bcl-2 and c-erbB-2 proteins are involved in the regulation of VEGF and of thymidine phosphorylase angiogenic activity in non-small-cell lung cancer

Tumour angiogenesis has been recently recognised as one of the most important prognostic factors in lung cancer. Although a variety of angiogenic factors have been identified, the angiogenesis process remains poorly understood. Bcl-2, c-erbB-2 and p53 are well-known oncogenes involved in non- small-cell lung cancer pathogenesis. A direct correlation of thymidine phosphorylase (TP) and of vascular endothelial growth factor (VEGF) with intratumoural angiogenesis has been reported. In the present study we investigated the possible regulatory role if bcl-2, c-erB-2 proteins in angiogenesis and in VEGF and TP expression in non-small-cell lung cancer. Two hundred sixteen specimens from T1,2-NO, 1 staged patients treated with surgery alone were immunohistochemically examined. Bcl-2 and c-erbB-2 were significantly inversely related to each other (P = 0.04) and both were inversely associated with microvessel density (P < 0.02). High TP and VEGF reactivity was statistically related to loss of bcl-2 expression (P < 0.01). A significant co-expression of c-erbB-2 with TP was noted (P = 0.01). However, TP expression was related to high angiogenesis only in cases with absence of c-erB-2 expression (P < 0.0001). c-erbB-2 expression in poorly vascularised tumours was linked with poor outcome (P = 0.03). The present study provides strong evidence that the bcl-2 gene has a suppressive function over genes involved in both angiogenesis (VEGF and TP) and cell migration (c- erbB-2) in NSCLC. TP and c-erbB-2 proteins are significantly, and often simultaneously, expressed in bcl-2 negative cases. However, expression of the c-erbB-2 abolishes the TP-related angiogenic activity. Whether this is a result of a direct activity of the c-erbB-2 protein or a consequence of a c- erbB-2-related immune response remains to be further investigated.

Expression and prognostic significance of a panel of tissue hypoxia markers in head-and-neck squamous cell carcinomas

Purpose: To investigate the expression pattern of hypoxia-induced proteins identified as being involved in malignant progression of head-and-neck squamous cell carcinoma (HNSCC) and to determine their relationship to tumor pO 2 and prognosis. Methods and Materials: We performed immunohistochemical staining of hypoxia-induced proteins (carbonic anhydrase IX [CA IX], BNIP3L, connective tissue growth factor, osteopontin, ephrin A1, hypoxia inducible gene-2, dihydrofolate reductase, galectin-1, IκB kinase β, and lysyl oxidase) on tumor tissue arrays of 101 HNSCC patients with pretreatment pO 2 measurements. Analysis of variance and Fisher's exact tests were used to evaluate the relationship between marker expression, tumor pO 2, and CA IX staining. Cox proportional hazard model and log-rank tests were used to determine the relationship between markers and prognosis. Results: Osteopontin expression correlated with tumor pO 2 (Eppendorf measurements) (p = 0.04). However, there was a strong correlation between lysyl oxidase, ephrin A1, and galectin-1 and CA IX staining. These markers also predicted for cancer-specific survival and overall survival on univariate analysis. A hypoxia score of 0-5 was assigned to each patient, on the basis of the presence of strong staining for these markers, whereby a higher score signifies increased marker expression. On multivariate analysis, increasing hypoxia score was an independent prognostic factor for cancer-specific survival (p = 0.015) and was borderline significant for overall survival (p = 0.057) when adjusted for other independent predictors of outcomes (hemoglobin and age). Conclusions: We identified a panel of hypoxia-related tissue markers that correlates with treatment outcomes in HNSCC. Validation of these markers will be needed to determine their utility in identifying patients for hypoxia-targeted therapy. © 2007 Elsevier Inc. All rights reserved.

The role of chemotherapy in the treatment of adult soft tissue sarcomas

Adult soft tissue sarcomas are relatively rare tumours which are curable with radical surgery. Approximately 50% of patients will develop inoperable disease or metastases for which chemotherapy may be inappropriate. Only two cytotoxic agents - doxorubicin and ifosfamide - have activity in > 20% of patients. For both these agents there is evidence of a dose-response relationship. There is currently no good evidence that combination chemotherapy confers a clinical benefit compared with single agents. Outside a clinical trial, standard first-line therapy should be with single agent doxorubicin at a dose intensity ≥ 70 mg2 every 3 weeks. Approximately 25% of patients may be expected to respond to this regimen. There is the suggestion that responses may occur to ifosfamide in patients who progress on doxorubicin. The role of chemotherapy in the adjuvant setting remains uncertain. Several trials have suggested a modest relapse-free and overall survival benefit for the use of post-operative chemotherapy and a recent overview of 14 randomised trials confirms a small though significant benefit. These benefits have to be weighed against the toxicity of chemotherapy. The importance of treating all patients with soft tissue sarcomas in clinical trials is stressed. There is an urgent need to define new active agents to treat this disease.

Prognostic value of TP53, KRAS and EGFR mutations in nonsmall cell lung cancer : the EUELC cohort

Nonsmall cell lung cancer samples from the European Early Lung Cancer biobank were analysed to assess the prognostic significance of mutations in the TP53, KRAS and EGFR genes. The series included 11 never-smokers, 86 former smokers, 152 current smokers and one patient without informed smoking status. There were 110 squamous cell carcinomas (SCCs), 133 adenocarcinomas (ADCs) and seven large cell carcinomas or mixed histologies. Expression of p53 was analysed by immunohistochemistry. DNA was extracted from frozen tumour tissues. TP53 mutations were detected in 48.8% of cases and were more frequent among SCCs than ADCs (p<0.0001). TP53 mutation status was not associated with prognosis. G to T transversions, known to be associated with smoking, were marginally more common among patients who developed a second primary lung cancer or recurrence/metastasis (progressive disease). EGFR mutations were almost exclusively found in never-smoking females (p=0.0067). KRAS mutations were detected in 18.5% of cases, mainly ADC (p<0.0001), and showed a tendency toward association with progressive disease status. These results suggest that mutations are good markers of different aetiologies and histopathological forms of lung cancers but have little prognostic value, with the exception of KRAS mutation, which may have a prognostic value in ADC. Copyright©ERS 2012.

Bioavailability study of oral and intravenous OGT 719, a novel nucleoside analogue with preferential activity in the liver

Purpose: Although oral fluoropyrimidine pro-drugs are increasingly being administered in preference to intravenous nucleoside analogues in cancer chemotherapy, their activation in malignant liver tissue may be insufficient. OGT 719 (1-galactopyranosyl-5-fluorouracil) is a novel nucleoside analogue, preferentially localized in hepatocytes and hepatoma cells via the asialoglycoprotein receptor. The aim of this study was to assess the systemic bioavailability of this rationally designed drug in 16 patients with advanced solid cancers. Method: Crossover pharmacokinetic study of oral (400 or 800 mg) and intravenous (250 mg/m 2) OGT 719. Results: Linear pharmacokinetics and oral bioavailability of approximately 25% were observed at the dose levels used in this study. Like other 5-FU prodrugs, considerable interpatient variability was observed in bioavailability following oral dosing. The mean half-life for oral doses was 4 h. OGT 719 was well tolerated. No objective tumour responses were demonstrated. Conclusion: The systemic bioavailability and half-life of oral OGT 719 are sufficient to merit dose escalation studies with frequent daily dosing. Subsequent efficacy studies should be performed in patients with primary and secondary liver malignancies.

Bioavailability study of oral and intravenous OGT 719, a novel nucleoside analogue with preferential activity in the liver

Purpose: Although oral fluoropyrimidine pro-drugs are increasingly being administered in preference to intravenous nucleoside analogues in cancer chemotherapy, their activation in malignant liver tissue may be insufficient. OGT 719 (1-galactopyranosyl-5-fluorouracil) is a novel nucleoside analogue, preferentially localized in hepatocytes and hepatoma cells via the asialoglycoprotein receptor. The aim of this study was to assess the systemic bioavailability of this rationally designed drug in 16 patients with advanced solid cancers. Method: Crossover pharmacokinetic study of oral (400 or 800 mg) and intravenous (250 mg/m 2) OGT 719. Results: Linear pharmacokinetics and oral bioavailability of approximately 25% were observed at the dose levels used in this study. Like other 5-FU prodrugs, considerable interpatient variability was observed in bioavailability following oral dosing. The mean half-life for oral doses was 4 h. OGT 719 was well tolerated. No objective tumour responses were demonstrated. Conclusion: The systemic bioavailability and half-life of oral OGT 719 are sufficient to merit dose escalation studies with frequent daily dosing. Subsequent efficacy studies should be performed in patients with primary and secondary liver malignancies.